THE ROLE OF THE INTESTINE IN CALCIUM HOMEOSTASIS IN THE LAYING HEN ( 1 )

THE ROLE OF THE INTESTINE IN CALCIUM HOMEOSTASIS IN THE LAYING HEN ( 1 )

S. HURWITZ Division of Poultry Science,

The Volcani Institute of Agricultural Research, ^Rehovot (Israel)

The metabolic

pathways

of calcium were

diagramatically

^described

by

^BRONNER

and AUBERT

( 19 65)

^{for the rat. In} this scheme

(fig. s),

^the various kinetic

parameters

as well as the various

pools

^{are defined}

mathematically

^but may be

assigned physio- logical

entities. The

pool (E l )

includes blood and soft tissue calcium and ^some bone calcium. It

exchanges,

^{with no net}

change

^{in mass with}

compartment E,

^{which is}

mostly

located in bone. The other

symbols represent

^{vectorial movements of calcium}

in and out of the

pool.

^{Calcium moves into the}

pool by

intestinal

absorption (V a )

^and

bone

resorption (V o -),

^{and out of the}

pool by

bone accretion

(V o+ ), urinary

^excretion

(V

u

)

^and

endogenous

^excretion

(V¡).

^In

laying hens,

^the

system

is further

compli-

cated

by

exits into the

yolk,

^{and most}

importantly

^{into the} egg shell.

In order to estimate the various

parameters

of calcium metabolism a tracer level of

calcium- 45

^is

injected intravenously,

and results of chemical and

radio-assays

^are

used in the mathematical solution of this model.

The solution of a similar model in the

laying

^hen is difficult due to several fac- tors :

a)

the existence of a

slowly exchangeable

calcium fraction in the

plasma

^{of this}

animal

(HuRmTZ, zg68), b) rapid

^{bone turnover with}

possible

^{return of}

calcium- 45

from bone to

circulation, c)

the size of

E l

^which may

change

^{with the}

laying cycle,

as reflected

by

^the

change

ⁱⁿ

plasma

^calcium

(H!RT!r,!NDY

^and

^{T AYLOR ,} 10 6 1 ) d)

^shell

formation,

^which

represents

^the

greatest

drain of calcium and is a discontinuous process.

The

laying cycle

ⁱⁿ the hen is

completed

in about 25-3o

hours,

^{out of which} egg shell is secreted

during

19-20 hours. Since shell secretion

occupies only part

^{of the}

laying cycle, large

^metabolic

changes

^{must take}

place

^{in the hen}

during

^{the commen-}

cement and termination of this _process. Such

changes

include the mobilization of

large

^amounts of calcium and

probably

^some

respiratory changes

^{needed in order to}

provide

^the

large supply

^of

^{CO 2}

^{to the}

forming

^shell.

Theoretically,

^this

special

^{need for calcium may be satisfied from the}

following

sources :

a) pool calcium, b)

decrease in

urinary

calcium excretion

c)

^{excess of bone}

resorption

^over bone formation

(net

^bone

catabolism)

^and

d)

increase in calcium

absorption.

CHANGES IN POOL SIZE

HERT!r,!!Dy and TAYLOR

( 3 ) reported

^a decrease in

plasma

^calcium

during

shell formation with hens fed a 2p. ¹⁰⁰calcium diet. Even if this observation holds under

optimal

^calcium

nutriture,

^{the amount} of calcium that _may be

gained

^from

such

change

^is in the range of a few

milligrams compared

^{to about 2} grams calcium needed for shell formation.

DECREASE IN URINARY CAI,CIUM EXCRETION

Several years ago ^we measured the rate of

urinary

^{excretion of} calcium in colos- tomized birds

(H URWITZ

^and

^GxlMmG!x, ig6i).

The balance sheet of this

experiment

is

given

^{in table i.}

Urinary

^calcium

excretion, although large

^{relative to} the size of the

hen,

^amounts

to

only

¹⁰ p. ¹⁰⁰ of the absorbed calcium.

Any

reduction in this excretion

during

shell formation can spare

only

^{a small} fraction of the shell calcium. Such reduction has been

recently reported by

^TAYLOR and Kixx!,!Y

(ig6 7 ),

^{but it} amounts to less per

day

^{than 100} mg.

UTILIZATION OP’ BONE CALCIUM

The

importance

^{of bone as a source of} calcium for the _egg

shell,

^{is well}

recognized.

Bone calcium

undergoes

^a contant turnover in which some of it is removed

(V o -)

and some is added to it

(V o+ ).

The calcium

leaving

^bone

by

the former process, ^enters and mixes in the

pool,

^{and can be}

secreted,

^as

part

^{of the}

pool-calcium,

^{into the} egg shell. When the hen is in calcium balance

(steady

^state

conditions),

the bone neither looses nor

gains

^any calcium. The calcium derived from bone is

deposited

^{in the} egg shell without net calcium losses to the bone.

Thus,

this bone calcium which reaches the shell does not have _any

physiological significance.

On the other

hand,

^{when the animal is}

deprived

^of

calcium,

^{the skeleton can}

supply practically

all the calcium needed for the _egg shell. This _process of net cal- cium removal from

bone,

^{which can continue until the animal is}

depleted

up ^to about 30

p. ¹⁰⁰ of its

body calcium,

^{is well} documented

(T AYLOR

^and

^{M OOR E,} r 95 6 ;

^HUR-

W I T

Z and

BAR, i966).

From calculations based on balance

data,

^TAYLOR

(ig6i) attempted

^{to show}

that

during

^shell

formation,

the animal must draw _upon its bone reserves. This cal-

culation, using

^{the data}

given

^{in table I, is as} follows : The average retention

(from

a

7 -day

^balance

period)

^was

i.8 3 g/ 24

^{hours or}

7 6 mg/hour.

Since each shell contains 2

.

05 g

calcium,

and is secreted

during

^{about 20}

hours,

^{the rate} of calcium

deposition

in the shell will be 102

mg/hour.

^It then follows that

during periods

^{of shell formation}

the hens are 26

mg/hour

^{short on} their calcium

supply,

^{and that this extra calcium}

must come from the skeleton. The calcium lost from the skeleton

during

^{shell for-}

mation will be

repleted

^{when no shell is}

formed,

^{and at} the end of the

cycle,

^{the hen}

will be in calcium balance.

This calculation is based on the

assumption

^{that the rate} of calcium

absorption

does not

change during

^the

laying cycle.

^It will be shown below that this

assumption

is not valid.

Furthermore,

^{if this} calculation is correct, ^{it must be}

possible

^{to detect} quan- titative

changes

^{in bone calcium}

during

^the

laying cycle.

^{In a}

study

^{in which we inves-}

tigated

the kinetic behaviour of

calcium- 45

^{in the}

laying

^hen

(H URWITZ , 19 6 4 ),

^we

also obtained some data on bone

composition.

^{In this}

experiment,

^{we were unable}

to show _any

changes

in the calcium content of

medullary

^{as well as} structural

bone, during

^the

laying cycle.

^{It should be}

emphasized

that the birds used in this

study

were fed

during

^their

^laying period

^diets

containing

^{3.5-4.o p. 100calcium.}

The above

findings

raised the

questions

of the entire

validity

^{of TaY!,oR’s calcu-}

lation. As no net

changes

in bone calcium could be

detected,

and since the skeleton is the

only large enough

reservoir of calcium in the

body,

^an increase in intestinal

absorption

^of calcium remained the

only

^means

by

^{which the animal} could obtain the amount of calcium it needed.

VARIATIONS IN INTESTINAL CALCIUM ^ABSORPTION

The classical balance

technique

^{has been}

widely

^{used to}

study

^{the calcium reten-}

tion in the

laying

^hen. Some studies ^were conducted with colostomized animals in order to measure the

apparent

^calcium

absorption

^{which is about 10} p. ¹⁰⁰

larger

than retention.

Due to the

delay

of about 6 hours between

ingestion

and excretion it is

impos-

sible to use balance

techniques

for studies of short duration. Such

techniques

^are

suitable for

long

^term

experiments

in which the differences between the intakes at the

beginning

and end of the balance trial become

insignifiant. Therefore,

the balance method could not be used to

study

the variations in calcium

absorption during

^the

laying cycle.

^{The use of an} unabsorbed reference

substance,

^{for this} purpose, seemed most

appropriate.

^Our

study

^involved

yttrium-gi

^as the reference substance

(H UR -

WITZ and

BAR, ig65).

Two groups of hens ^were fed diets

containing, respectively,

i.go and

3 . 5 6

p. ¹⁰⁰ calcium. Both diets were

uniformly

labelled with

yttrium- 9 1.

After four

days

^{on the}

respective diets,

^{the hens were} killed and their intestine

separated

^into various seg- ments, ^{the contents of which were}

analyzed

for calcium and

yttrium- 9 1.

^The

apparent absorption

^at any level of the intestine were calculated from

Ca/Y-gi

^ratio.

From each group of

hens,

^{some were killed}

during early

^shell

calcification,

^some

during

late shell calcification and

others,

^{when no shell was formed.}

The overall

absorption

calculated from the

Ca/Y-gi

^{ratio at the distal}

^intestine,

is

given

^{in table 2.}

Although percentage absorption

^{was somewhat}

higher

^{for hens}

receiving

^the

lower calcium

diet,

this difference was not

significant,

^the

changes

^{in calcium}

absorp- tion,

^{due to} shell formation ^are rather

striking,

^with

absorption

almost doubled.

There was little difference between

periods

^of

early

and late calcification. More recent work of TAYLOR and associates

( 19 6 7 )

also found the

absorption

of calcium

greater

in

laying days

^as

compared

^{to non}

laying days.

The

percentage absorption

^of

7 o-8o

p. ¹⁰⁰ may be considered

maximal,

^since

even at much lower levels of

dietary

^{calcium it would}

rearely

^{exceed this limit.}

If one assumes a constant

supply

of nutrients into the

digestive

^tract

(according

to our

experience

this situation is

approached),

^{one can} calculate the rate of calcium

absorption during

shell formation. This ^rate is ₁₀₄ and

5 8

mg per

hour,

^{for the}

3!56

p. ¹⁰⁰and the i.go p. ¹⁰⁰calcium

diets, respectively.

^{For the hens}

receiving

^the

high

calcium diet the calcium absorbed is sufficient ^{to meet} the

need,

^{if the rate of} shell calcification is constant, but this is not the case with those

receiving

^{the low}

calcium diet.

This

experiment explicitely

^shows that when the calcium intake is

suihcient,

and continues

during periods

^{of shell}

formation,

^{there is} little need for bone to con-

tribute any net amount of calcium to the _egg shell. The intestine has

proved

^{to be}

of utmost

importance

in the calcium homeostasis of the

laying

^hen.

It is evident that the

moderately

^low calcium diet of z.go p. ^{100 was} insufficient to enhance

absorption

above what we termed above the maximum

percentage absorp-

tion.

Undoubtedly,

^{the birds}

receiving

this diet had been in

negative

^calcium

balance,

and would have returned to balance

by

reduction in shell secretion and rate of _egg

production (HuRmTZ

^and

G RIMIN GE R , 10 6 0 ).

^{It was,}

therefore,

^of

importance

^{to see}

whether the intestine reacts more

rapidly

^{to a}

challenge

^{of an acute calcium}

deficiency.

EFFECT OF ACUTE DEPLETION ^ON CALCIUM ABSORPTION

The classical balance method was sufficient for

studying

^this

problem

^{since we}

did not look for hour to hour

changes

ⁱⁿ

absorption (H URWITZ

^and

BAR, ig66).

Two _groups of hens were fed for 6

days (control period)

^diets

containing

^1.82

and 3.92 p. ¹⁰⁰

calcium, respectively.

This control

period

^{was followed}

by

^a

2 -day depletion period during

which all birds received a « calcium-free » diet

(o.i2

^{p. 100}

calcium). Lastly,

^{the hens were returned to their}

original

^{diets for} additional 6

days (repletion period).

Calcium balances were conducted

during

the control and

repletion periods.

Results of this

experiment (table 3 )

^{indicate that} hens of both groups

responded

to the

challenge

of calcium

depletion by

^a substantial increase in their calcium

absorp-

tion.

The combination of an increase in

absorption

with the decrease in shell

secretion,

enabled the birds to maintain a

positive

calcium balance

during repletion,

^thus

reple- ting

their skeleton. In the 1.82 _p. 100

calcium-birds,

^the

absorption

of calcium had not

immediately

^reached the maximal

value, although they

^{were in a distinct} nega- tive balance. This would

suggest

^that the response of the intestine to the

challenge

of calcium

depletion

^{is to some}

degree proportional

^{to the}

magnitude

^{of the}

challenge.

THE MECHANISM OP’ REGULATION ^OF’ CALCIUM ABSORPTION

We have demonstrated the role of the intestine in calcium homeostasis in two instances : a response ^{to a} stimulus of an increase in the calcium need due to shell

formation,

^{and a} response ^{to a}

challenge

^{of calcium}

deprivation.

^We

^have,

^however

no indication if the mechanism of this _response is the same or different in those two instances.

K IMBERG

el al.

( 19 6 T )

showed that ^« active

transport

» of calcium

developed

in intestinal

segments

^{lower than} the duodenum in rats

subject

^{to a} low calcium

regime.

This conclusion was based on in vitro studies. !I!DDama and BRONNER

( 19 68)

showed that the appearance of calcium

binding protein (CaBP)

ⁱⁿ the intestinal mu- cosa was an inverse function of

dietary calcium,

^and associated its appearance with the action of the

parathyroids.

^{It is most}

tempting

^to

extrapolate

^these

findings

^to

the

regulation

^{of calcium}

absorption by

^the

laying

hen-intestine.

However,

^before

doing

^{so one must evaluate} the mechanism of calcium

absorption

^{in the} hen : is it

uphill

^or

^downhill,

and if the

latter,

^{is it}

simple

^or facilitated diffusion.

After

developing

^{an in vitro} system ^for

laying

^hen

intestine,

^Mr. BAR, ^{in our}

laboratory, investigated

the mechanism of the mucosal

transport

of calcium

(BAR

and HURWITZ,

r 9 6 9 ).

On the basis of kinetic evidence and the use of certain inhibi- tors, ^{it was} concluded that the mucosal

uptake

^{of calcium was a}

simple

^diffusion

process. The ^mucosal

uptake

^{can be} considered the first

step

ⁱⁿ

absorption,

^{and there-}

fore a mechanism other than

simple

^diffusion may still exist for the

subsequent

^trans-

port

of calcium. In an effort to

study

the transmural

transport

^{of calcium in the}

laying

hen

intestine,

^{we tried}

gut loops,

^a

preparation

^{similar to} that used with rat intestine

(Krn IS

xRT

^et

^al., ig6i). Unfortunately,

^the

laying

^{hen intestine was found to}

be imper-

meable ^to

calcium,

^{under in} vitro conditions.

The other more difficult

possibility

^{was to}

study

the calcium

transport

^{in vivo}

and relate it to the

corresponding driving

^forces

operating

^on intestinal calcium.

The first

study

^{of this}

type

^{has been}

recently reported (H URWITZ

^{and BAR,}

i

9

68).

^This

^study

^involved measurements of luminal calcium

activity,

^{and trans-}

mural electrical

potential,

^{as well as} estimation of the net calcium

absorption, using yttrium-gi.

The results of this

study,

^{as well as other}

experiments,

^{tend to}

suggest simple

^{diffusion as the mechanism of calcium}

absorption,

ⁱⁿ

^vivo,

^{in the}

laying

^hen.

However, considerable more evidence is needed to

support

this conclusion.

As mentioned

above, regulation

of calcium

absorption

^{can be}

explained readily

if active

transport

^{and a calcium carrier are evoked. But} if calcium is

transported by simple diffusion,

^how could it be

physiologically

controlled ? HARRISONand HARRISON

( 19

65) ^clearly

^{showed mucosa to be a}

permeability

barrier for calcium. SCHACHTER

( 19 6 3

)

^{showed that this}

permeability

barrier in the rat

jejunum

^{and ileum was} oxygen-

dependent.

^{It thus seems}

possible

^that

by

^{some metabolic}

change,

^the

permeability

of the mucosa for calcium can be

modified,

^thus

effecting

^the

regulation

of intestinal

absorption.

SUMMARY

In the laying hen, calcium intestinal absorption ^increases during egg shell formation. On account of this increase, skeleton calcium input in egg shell is poor. ^I

Calcium absorption also increases when the animal is fed a diet

poor

in calcium.

We may conclude that in the laying ^{hen, intestine} plays ^an important part in calcic homeos- tasis. The possible ^{cause of this} regulation is discussed.

RÉSUME

RÔLE DE L’INTESTIN DANS ^« L’HOMÉOSTASIE CALCIQUE ^»

DE LA POULE PONDEUSE

L’absorption intestinale du calcium augmente ^{chez la} poule pondeuse pendant la formation de la coquille de l’oeuf. Du fait de cette augmentation, la contribution ^en calcium du squelette

pour la coquille ^{de l’oeuf est faible.}

L’absorption du calcium augmente également lorsque ^l’animal reçoit ^un régime ^{carencé en}

calcium.

Il en est conclu que l’intestin joue ^{un rôle} important dans l’homéostasie calcique ^{de la} poule pondeuse. ^{La cause} possible ^{de cette} régulation ^{est discutée.}

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BRONNER F., AUBERT J.-P., 1965. ^Bone metabolism and regulation of the blood calcium level in rats.

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